Humanize

This tab provides an alternative view of unusual residues and residue distributions to aid in humanization, de-immunization and optimization studies.

The heatmap display

The main display shows residue frequencies as a 'heatmap'. Each horizontal position represents an amino acid along the sequence while each vertical column can be thought of as looking down on the relevant distribution graph for the Target Organism seeing high peaks (common residues) in green, medium peaks in yellow and low peaks (unusual residues) in red.

The CDRs (by default the Kabat definition) are indicated with slightly darker colours.

The first row displays the sequence of interest coloured by reference to the Source Organism. Typically you would set this to your donor species (e.g. mouse).

The second row (and the heatmap below it) colours residues based on the distributions seen in the Target Organism. Normally you would set this to your acceptor species (typically human).

The next 20 rows are the main heatmap with distributions derived from the Target Organism. Common residues are shown in green; unusual residues are shown in red; other residues are shown in yellow.

The final row starts out as the same as the second row - i.e. the sequence of interest coloured by reference to the Source Organism.

Hovering over any residue in either of the top two rows will display a popup giving information about that residue including the residue number in each of the numbering schemes.

Clicking any residue in either of the top two rows will update the Distribution display in the bottom right hand corner of the page. (See below.)

Hovering over a cell in the heatmap will display a popup indicating the mutation from the sequence of interest that is represented by this cell.

Clicking on a residue in the heatmap will introduce the mutation to the sequence at the bottom of the heatmap, updating the colour in that sequence appropriately.

Display controls

The Source Organism dropdown allows the organism to be selected which is used for colouring the sequence in the first row of the heatmap table.

The Target Organism dropdown allows the organism to be selected which is used for colouring the sequence in the second row of the heatmap table and in the main heatmap below it.

The Highlight CDRs dropdown allows different definitions of the CDRs to be selected for highlighting

The Scaled / Not Scaled radio-buttons select whether the colouring of the heat map uses the raw percentage occurrence (Not Scaled) or the percentage occurrence with respect to the most common residue at that position (Scaled). For a particular position, if all residues are used equally then, without scaling, they would all have a frequency of 0.05; with scaling they would all have a frequency of 1.0.

The Lower limit / Upper limit boxes and associated two-handle slider allows you to specify the colouring thresholds. Residues having a frequency above the Upper limit will be green; residues having a frequency below the Lowe limit will be red; residues having a frequency between the two limits will be yellow.

The Export button allows the modified sequence to be exported. It appears in a popup in FASTA format that can be cut-and-pasted into another application.

The Reset button will change all mutated residues back to the original sequence.

Distributions

A bar chart of the residue distribution for the selected residue is displayed. The residue present in the sequence of interest is highlighted with a downwards arrow at the top of the bar. To the right is an associated table containing the residue counts in non-identical sequences and the relative frequencies. You may also download a version of the graph and a CSV file of the data.

Click the residues in the top two rows of the main heatmap table to change the distribution that is displayed in the graph.